Ionic channels of cell membranes are the basic sites where ionic fluxes take place. The modern era of the study of drug-channel interactions began when voltage clamp techniques were used to demonstrate the block of Sodium, (Na.sup.+), and potassium, (K.sup.+), channels of squid axons caused by procaine and cocaine. Narahashi, Ann Neurology (1984); 16(suppl): S39-S51.
This invention concerns proteins which regulate or constitute the pore region of potassium channels. Potassium channels appear to be ubiquitous, found even in bacteria. See, R. Milkman, "An Escherichia Coli homologue of eukaryotic potassium channel proteins" Proc. Natl. Acad. Sci. USA, Vol 91, pp. 3510-3514, (1994). Pharmacological, biophysical and molecular studies have revealed multiple subtypes for membrane ion channels that form potassium selective pores in the plasma membrane of many mammalian cells. One method of classifying K channels is based on what regulates channel activity or function. For example, one class can be defined as K channels modulated by transmembrane voltage, another class modulated solely by calcium and/or nucleotides, and yet a third class modulated by G protein involvement. However, in a more simplistic manner, one can classify the family of K channels simply by their respective gating properties. In other words, a comparison of the pharmacological and electrophysiological properties of potassium channels has given rise to an operational definition for grouping the various subtypes based largely on their gating properties. At present, potassium channels of known amino acid sequence comprise two distantly related protein families. One of these channel families is termed, "voltage-gated," the other channel family is termed "inward rectifying."
The structure of the voltage-gated channel protein is known to be comprised of six membrane spanning domains in each subunit, each of which is regulated by changes in membrane potential. B. Hille. "Ionic Channels of Excitable Membranes" (Sinauer, Sunderland, Mass., 1992). Voltage-gated potassium channels sense changes in membrane potential and move potassium ions in response to this alteration in the cell membrane potential. Molecular cloning studies on potassium channel proteins has yielded information primarily for members of the voltage-gated family of potassium channels. Various genes encoding these voltage-gated family of potassium channel proteins have been cloned using Drosophila genes derived from both the Shaker, Shaw and Shab loci; Wei, A. et. al., Science (1990) Vol. 248 pp. 599-603.
Unlike the voltage-gated channel proteins with six membrane spanning regions, the inward rectifier channels have only two membrane spanning domains, each sensitive to changes in the net potassium concentration. Within this class of channels are the ATP-sensitive potassium channels. These channels are classified by their sensitivity to concentration fluxes in ATP. The ATP-sensitive, or ATP-gated, potassium channel is an important class of channels that links the bioenergetic situation of the cell to changes in cell function. These channels are blocked by high intracellular ATP concentrations and are open when ATP decreases. Lazdunski (1992); M. Lazdunski et al., "ATP-Sensitive K.sup.+ Channels", Renal Physiol. Biochem. Vol. 17: pp. 118-120 (1994).
Although ATP-gated potassium channels were originally described in cardiac tissue; Noma, A. Nature (1983) Vol. 305 pp. 147-148, they have subsequently been described in pancreatic .beta.-cells; Cook et. al., Nature (1984) Vol. 311 pp. 271-273, vascular smooth muscle; Nelson, M. T. et. al., Am. J. Physiol. (1990) Vol. 259 pp. C3-C18 and in the thick ascending limb of the kidney; Wang, W. et. al. Am. J. Physiol. (1990) Vol. 258, pp. F244-F-253.
The ATP-sensitive, or ATP-gated potassium channels play an important role in human physiology. The ATP-sensitive potassium channel, like other potassium channels, selectively regulate the cell's permeability to potassium ions. These channels function to regulate the contraction and relaxation of the smooth muscle by opening or closing the channels in response to the modulation of receptors or potentials on the cell membrane. When ATP-sensitive potassium channels are opened, the increased permeability of the cell membrane allows more potassium ions to migrate outwardly so that the membrane potential shifts toward more negative values. When the membrane potential shifts toward more negative values the opening of the voltage-dependent calcium channels is reduced, this reduces the influx of calcium ions into the cell because the calcium channels become "increasingly less open" as the membrane potential becomes more negative. Consequently, drugs having ATP-sensitive potassium channel opening activity, drugs known as potassium channel openers, can relax vascular smooth muscle and are useful as hypotensive and coronary vasodilating agents. In contrast, drugs having ATP-sensitive potassium channel blocking activity, drugs known as potassium channel blockers, inhibit ATP-sensitive potassium channels by decreasing potassium efflux, leading to membrane depolarization which opens voltage-gated Ca.sup.2+ channels. Arkhammar et al. (1987) "Inhibition of ATP-regulated K.sup.+ channels precedes depolarization-induced increase in cytoplasmic free Ca.sup.2+ concentration in pancreatic B-cells", J. Biol. Chem. 262: 5448-5454. These drugs find optimal use in the stimulation of insulin secretion in type II diabetes mellitus.
A relatively large number of compounds are now known which open cell membrane ATP-sensitive potassium channels, particularly in smooth muscle: minoxidil sulfate, diazoxide and nicorandil are well known potassium channel openers. The target site for these agents is presumably on the potassium channel itself, but may also be on an associated regulatory protein. Isolation of the target site for the potassium channel openers would allow for protein sequence analysis and cloning of those potassium channel opener proteins. Similar analyses of drug binding proteins in K.sub.ATP channels have been performed for the class of K channel blockers such as glyburide. Sulfonylurea receptors have been analyzed on a variety of cell and tissue types using a photoactivable form of glyburide. Aguilar-Bryan, L., et al., "Photoaffinity Labeling and Partial Purification of the B Cell Sulfonylurea Receptor Using a Novel, Biologically Active Glyburide Analog", J. Biol. Chem. (15 May 1990) Vol. 265, pp. 8218-8224.
Potassium channel openers represent a widely diverse series of compounds which all have the reported effect of opening only a subset of channels described as sensitive to ATP. As explained above, these compounds cause physiological responses by increasing membrane permeability to potassium, this leads to hyperpolarization of the cell membrane and temporal desensitization to electrical and chemical stimuli.
Openers which target these channels have been synthesized as possible drugs in hypertension, angina pectoris, coronary heart disease, asthma, and urinary incontinence. Blockers which target these channels include the sulfonylureas, such as glyburide. The latter is an example of an important drug which targets K.sub.ATP channels in the pancreas, thus providing a treatment for non-insulin dependent diabetes mellitus.
The rationale for the effectiveness of these drugs in targeting the K.sub.ATP channel resides in the fact that this channel constitutes the main resting conductance in the B-cell. Depolarization of the channel by the sulfonylurea blockers ultimately results in insulin release.
Despite the apparent selectivity afforded by such drugs, it also appears true that openers have multiple effects on target cells as well as selective effects on several tissue types. K Lawson and P. E. Hicks, "Potassium Channel Openers: Pharmacological Anomalies Suggest Heterogeneous Sites of Action", (1993) Curr. Opin. Invest. Drugs Vol 2 pp. 1209-1216. It is the latter effect, that of multiple tissue targeting, that has reduced the importance of the K channel openers as selective marketable drugs. It is essential to understand what confers selectivity of drugs to specific organs before a systematic approach can be made towards drug design.
The membrane proteins which bind to potassium channel openers are believed to be structurally related, although it isn't clear whether drug selectivity is imparted by the channel protein itself or by the contribution of accessory proteins. These proteins, which bind to selective drugs, may be novel K channels or they may be one of several K channel accessory proteins that act in concert with the primary K channel protein and that are needed by the system for the proper physiological response.
An analogous system using the channel blocker, glyburide, has been explored for pancreatic B cell K.sub.ATP channels. Aguilar-Bryan, L., et al., "Co-Expression of Sulfonylurea Receptors and K.sub.ATP Channels in Hamster Insulinoma Tumor (HIT) Cells: Evidence for direct association of the receptor with the channel", J. Biol. Chem. (1992), Vol. 267 pp. 14934-14940.
This invention describes the isolation and identification of a new protein, p56, useful for the identification of selective drugs that will selectively open or close K channels. P56 is the first high affinity cyanoguanidine binding protein to be identified using a K channel opener photoactivable probe. Unexpectedly, this opener was shown to only bind to P56 in intact cells.